syntax tophat2 for reference index

Question

[marongiu.luigi]

Hello,

I want to use Tophat to align reading for RNA-seq analysis. I downloaded the sequences SRR390728_1.fastq and SRR390728_2.fastq, quality trimmed with

java -jar /usr/bin/trimmomatic PE SRR390728_1.fastq SRR390728_2.fastq paired1.fq unpaired_1.fq paired2.fq unpaired_2.fq SLIDINGWINDOW:4:20 MINLEN:20 ILLUMINACLIP:/usr/local/lib/Trimmomatic/adapters/TruSeq2-PE.fa:2:30:10:1:true

and then downloaded the reference human sequences Homo_sapiens.GRCh38.dna.toplevel.fa and Homo_sapiens.GRCh38.90.gtf, which I renamed Hsapiens_GRCh38.fa and Hsapiens_GRCh38.gtf respectively. I then indexed them with

bowtie2-build -f  Hsapiens_GRCh38.fa Hsapiens_GRCh38
tophat2 -G Hsapiens_GRCh38.gtf --transcriptome-index=Hsapiens_GRCh38.tr Hsapiens_GRCh38

and then copied everything in the same folder, so that the content of the folder in use is:

$ ls
align.sam               Hsapiens_GRCh38.rev.1.bt2l  SRR390728_2.fastq
Hsapiens_GRCh38.1.bt2l  Hsapiens_GRCh38.rev.2.bt2l 
Hsapiens_GRCh38.2.bt2l  Hsapiens_GRCh38.tr          
Hsapiens_GRCh38.3.bt2l  paired1.fq                  unpaired_1.fq
Hsapiens_GRCh38.4.bt2l  paired2.fq                  unpaired_2.fq
Hsapiens_GRCh38.fa      Hsapiens_GRCh38.gtf     SRR390728_1.fastq

with Hsapiens_GRCh38.tr being a folder.

Then I ran the alignment with:

$ tophat2 -o SRR390728_aln --transcriptome-index=Hsapiens_GRCh38.tr Hsapiens_GRCh38 paired1.fq paired2.fq

[2017-10-12 11:26:19] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2017-10-12 11:26:19] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2017-10-12 11:26:19] Checking for Bowtie index files (transcriptome)..
Error: Could not find Bowtie 2 index files Hsapiens_GRCh38.tr.*.bt2l)

Then I gave the index as a path adding './' and I got:

$ tophat2 -o SRR390728_aln --transcriptome-index=./Hsapiens_GRCh38.tr Hsapiens_GRCh38 paired1.fq paired2.fq
   [2017-10-12 11:27:36] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2017-10-12 11:27:36] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2017-10-12 11:27:37] Checking for Bowtie index files (genome)..
[2017-10-12 11:27:37] Checking for reference FASTA file
[2017-10-12 11:27:37] Generating SAM header for Hsapiens_GRCh38
Error: Opening file ./Hsapiens_GRCh38.tr.gff

The content of Hsapiens_GRCh38.tr is:

./Hsapiens_GRCh38.tr$ ls
Hsapiens_GRCh38.1.bt2  Hsapiens_GRCh38.fa         Hsapiens_GRCh38.rev.2.bt2
Hsapiens_GRCh38.2.bt2  Hsapiens_GRCh38.fa.tlst    Hsapiens_GRCh38.ver
Hsapiens_GRCh38.3.bt2  Hsapiens_GRCh38.gff
Hsapiens_GRCh38.4.bt2  Hsapiens_GRCh38.rev.1.bt2

My questions are:

1. Have I made an error in the syntax? (how Hsapiens_GRCh38.tr.gff came out?)

2. it is possible to give a path to the .tr folder and the other indices, so I can use a single folder for all the alignments?

Thank you

[GenoMax]

I want to use Tophat to align reading for RNA-seq analysis.

Only reason to continue using TopHat at this time is nostalgia.

There are much better/accurate tools that you should switch to. Even authors of TopHat have suggested using HISAT2 (their new tool). STAR, BBMap (any other splice aware aligner) are excellent other choices.

[marongiu.luigi]

I know, it is nostalgia indeed. I am already switching to STAR and HISAT, but I wanted to get to know Tophat for completion. The index was built a folder above Hsapiens_GRCh38.tr (actually the latter was created by Tophat).

[GenoMax]

The transcriptome index was built inside Hsapiens_GRCh38.tr folder? Are the missing files in there?

[marongiu.luigi]

The indexing looked done OK, there were no error messages. I provided the list of files in use; does not look to me there is something missing.

Answer 1

[e.rempel]

Ok. Let's start from the beginning. First, I would update Bowtie2 to the current version (2.3.3.1). You must check .gtf and .fasta files for compatibility (same chr names etc).

Bowtie index (hg38... files will be created)

bowtie2-build -f Hsapiens_GRCh38.fa hg38

The command for transcriptome index generation must be (creates Hsapiens_GRCh38...)

tophat2 -G Hsapiens_GRCh38.gtf --transcriptome-index=Hsapiens_GRCh38.tr/Hsapiens_GRCh38 hg38

TopHat2 run

tophat2 -o SRR390728_aln --transcriptome-index=Hsapiens_GRCh38.tr/Hsapiens_GRCh38 hg38 paired1.fq paired2.fq

[marongiu.luigi]

Thank you. I have upgraded Bowtie to 2.3.3.1 using the precompiled version for linux 64 bit; the version 2.2.9.0 I was using instead was generated from the source with make. However, while with the former version it took me overnight to generate the index, with the newer I had to stop it after 48 h because the process was still running. Is that normal? this slow mode is perhaps due to the precompiled version?

[marongiu.luigi]

It worked. I built bowtie2 2.3.3.1 from source and this time everything went smoothly. I could even use a fixed folder where I ma keeping the reference indices (/home/RefSeq/Human):

tophat2 -o SRR390728_aln --transcriptome-index=/home/RefSeq/Human/Hsapiens_GRCh38.tr/Hsapiens_GRCh38 /home/RefSeq/Human/hg38 paired1.fq paired2.fq

Answer 2

[e.rempel]

You should include the prefix Hsapiens_GRCh38 in the path to the index:

--transcriptome-index=Hsapiens_GRCh38.tr/Hsapiens_GRCh38

[marongiu.luigi]

I got the following:

$ tophat2 -o SRR390728_aln --transcriptome-index=Hsapiens_GRCh38.tr/Hsapiens_GRCh38 paired1.fq paired2.fq

[2017-10-12 16:05:10] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2017-10-12 16:05:10] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2017-10-12 16:05:10] Checking for Bowtie index files (transcriptome)..
[2017-10-12 16:05:10] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (paired1.fq.*.bt2l)

$ tophat2 -o SRR390728_aln --transcriptome-index=./Hsapiens_GRCh38.tr/Hsapiens_GRCh38 paired1.fq paired2.fq


[2017-10-12 16:05:32] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2017-10-12 16:05:32] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2017-10-12 16:05:32] Checking for Bowtie index files (transcriptome)..
[2017-10-12 16:05:32] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (paired1.fq.*.bt2l)

I reckon the base name 'Hsapiens_GRCh38' should really be on its own.

[marongiu.luigi]

I even tried to remove the .tr from the index name:

$ tophat2 -o SRR390728_aln --transcriptome-index=./Hsapiens_GRCh38 Hsapiens_GRCh38 paired1.fq paired2.fq

[2017-10-12 16:12:02] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2017-10-12 16:12:02] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2017-10-12 16:12:02] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (Hsapiens_GRCh38.*.bt2l)

Yet the *.bt2l files are in the current folder. Even by moving them into Hsapiens_GRCh38.tr, there is an error:

$ tophat2 -o SRR390728_aln --transcriptome-index=Hsapiens_GRCh38.tr Hsapiens_GRCh38 paired1.fq paired2.fq

[2017-10-12 16:16:32] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2017-10-12 16:16:32] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2017-10-12 16:16:32] Checking for Bowtie index files (transcriptome)..
Error: Could not find Bowtie 2 index files Hsapiens_GRCh38.tr.*.bt2l)
$ tophat2 -o SRR390728_aln --transcriptome-index=./Hsapiens_GRCh38.tr Hsapiens_GRCh38 paired1.fq paired2.fq

[2017-10-12 16:16:51] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2017-10-12 16:16:51] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2017-10-12 16:16:51] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (Hsapiens_GRCh38.*.bt2l)